Project Summary Psoriasis affects 2-3% of the US population. Currently, there are no high throughput screening methodologies to develop new psoriasis drugs. To meet this significant unmet pharmaceutical need, we will screen for drugs to treat this disease by using a 3D skin system established with 3D bioprinting, induced pluripotent stem cell (iPSC) reprograming and CRISPR genome-editing technologies. We successfully constructed 3D skin using exclusively iPSC-derived fibroblasts iFBs) and keratinocytes (iKCs), demonstrating the feasibility of this approach. 3D bioprinting can allow us to reproducibly construct complex structures to recapitulate human tissues/organs, and miniaturize the 3D fabricated skin for high throughput screening. Psoriasis is an inflammatory skin disorder with abnormal epidermal hyperproliferation, compromised barrier function, upregulated keratin 16 (KRT16), downregulated filaggrin (FLG) and excessive secretion of cytokines such as IL-8, which can be monitored with transepithelial/transendothelial electrical resistance (TEER), fluorescence reporters and IL-8 ELISA as readouts. We postulate that compounds will manifest their anti-psoriatic effects through the reduction in disease phenotypes that can be detected by relevant readouts. Psoriasis can be recapitulated and assessed using cytokine treatment on skin constructs derived from primary fibroblasts (FBs) and keratinocytes (KCs), however it is labor intensive and inefficient to produce large quantity of T cells and reporter-containing iPSC-derived keratinocytes (iKCs), Therefore, in this project, we will use cytokine treatment and skin constructs fabricated with primary cells for initial screening and then use iPSC-derived 3D skin containing T cells and reporters for subsequent compound validation, confirmation and the mechanism of actions (MOA) studies. Specifically, we will use FBs and KCs to bioprint 3D skin in a 96 well format, and treat with a cytokine cocktail containing TNF-a, IL-1a, IL6 and IL17A, for a pilot screen of 50 inflammation-related compounds to optimize the high throughput procedure that then will be applied to screen a library of 1000 compounds using TEER and IL-8 ELISA. Subsequently, we will validate the lead compounds using lactate dehydrogenase (LDH) toxicity assays and iPSC-derived skin constructs that are generated from healthy donor cells and containing GFP reporters for KRT16 and mCherry for FLG. Finally, bioprinted psoriasis-specific iPSC derived skin constructs containing reporters and Th17/Th1 cells will be used to confirm the lead hits and delineate the MOA of compounds using RNA-seq. Completion of this project will provide us with compounds that may be developed into drugs to treat psoriasis. Moreover, our 3D skin model-based high throughput system can be easily adapted to screen for drugs to treat other inflammatory skin diseases.